The goal of this project is to investigate the cellular and molecular mechanisms by which cytokines regulate inflammation and host anti-tumor immune responses in vivo, particularly as they relate to the complex cellular interactions between the tumor and organ microenvironments. Our first approach is to characterize the profile of tumor-infiltrating leukocytes during tumor progression. Using a transplantable, metastatic renal cell carcinoma, we are analyzing leukocytes contained within the primary tumors that develop in the kidney as well as those in the lung and livers, which serve as primary and secondary sites of tumor metastases, respectively. Chemokines are chemotactic cytokines that serve to recruit specific leukocyte subsets into regions of ongoing inflammatory responses. We are utilizing mice deficient in various chemokine receptors to identify the mechanisms whereby monocytes, T cells and other inflammatory cells are recruited to the tumor site. Once present at the developing tumor site, these cells are capable of producing many different soluble mediators, such as interferon gamma (IFNg), nitric oxide, and VEGF that may influence tumor progression. By regulating the ability of these cell types to accumulate within tumors, we are identifying the role that these cells play during both primary tumor progression and metastasis to distant organs. Our immunotherapeutic regimens include IL-2 or IL-15 in combination with agonistic antibody to CD40. After treating tumor-bearing mice with this combination, we have identified the recruitment of macrophages and T cells that appear to be critical mediators of anti-tumor responses. Our data have illustrated the potential for dramatic mechanistic differences in biological effects mediated by anti-CD40 alone versus its use in combination with IL-2 that includes the synergistic upregulation of IFNg and nitric oxide expression that controls tumor burden. To date, we have shown IL-2/aCD40 induces enhanced antitumor responses that depend on the infiltration of established tumors by effector CD8+ T cells and a concomitant IFNg-dependent reduction in CD4+/FoxP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC) and Th2 chemokine expression within the tumor-microenvironment. These results may help to explain the limited clinical efficacy of aCD40 as a single agent based on its inability to remove Tregs and MDSC specifically from within the tumor microenvironment and they suggest that anti-CD40 may be more beneficial in combination with other selected immune agents, such as IL-2. We have also translated our earlier preclinical findings using IL2 in combination with IL12 to human clinical trials. Our current and future studies seek to clarify the cellular and molecular events critical for the observed biological effects of aCD40, and the potential for complementary use of aCD40 with rationally selected molecularly targeted agents. In another approach, we have combined anti-CD40 with an ATP competitive mTOR drug, AZD8055, developed by AstraZeneca. The rationale for this strategy is two-fold. First, mTOR inhibition represents is currently a leading clinical target for RCC. We also hypothesized that combining AZD8055 with aCD40 antibody would induce more efficient antitumor effects by a combination of direct tumor killing and subsequent release of tumor-associated antigens to antigen presenting cells and coincident modulation of immune cell functions in vivo. The results of our recently published study show that in a syngeneic mouse metastatic renal cell carcinoma (RCC) model, AZD8055 and aCD40 synergize for tumor regression by activating macrophages and DCs and inducing strong Th1 immune responses in the tumor microenvironment. In another approach, we have begun to analyze the role that dendritic cells play during the anti-tumor responses achieved by immunotherapies. Treatment with IL-12, a potent immunoregulatory cytokine with dramatic anti-tumor effects on its own, increases dendritic cells differently in the liver as compared to lymphoid organs such as the spleen. We are currently analyzing whether therapies that combine IL-12 with other immunomodulatory cytokines such as IL-2 or IL-15 offer a more effective immune response against renal cell carcinoma by enhancing the antigen presenting capability of these important effector cells. Furthermore, there is dynamic cross-talk between dendritic cells and NK and NK-T cells and therapies that modulate the numbers or function of these cells may have significant impact upon the ability of DC to prime T cells for specific anti-tumor responses. In addition, we have identified a specific accumulation of T regulatory cells within the tumor microenvironment, which may serve to suppress anti-tumor responses. We have identified tumor necrosis factor alpha (TNFa), a critical proinflammatory cytokine, as possibly being involved in the recruitment and/or expansion of T regulatory cells. We are targeting this cytokine by molecular and biochemical approaches that may ultimately be useful in the clinic for counteracting the establishment of a suppressive tumor microenvironment. We have also identified a putative immunosuppressive role for NK-T cells and we are now developing immunotherapeutic regimens that reduce the numbers and activity of these cells in different organs. By characterizing the role of inflammation upon tumor progression, these overlapping approaches will hopefully lead to the development of immunotherapeutic approaches that skew the inflammatory response to counter tumor growth.